Daniel:Notebook/HiResChrPaint/2013-8-14: Difference between revisions
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>Djacobse (Created page with "=Amino Group Primers= Back to Calendar So the new primers are in. We ordered them a few weeks ago. All the primers have a 5' amino group,...") |
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So the new primers are in. We ordered them a few weeks ago. All the primers have a 5' amino group, which should be compatible with the Alexa-dye coupling system. Thanks to [[Matt:LabNotes/2013-9-25|Matt's results]] yesterday, I know that when the dye coupling does not work, the Centri-Sep columns yield very little dye. So, before going nuts on making new probes, I'm going to test the water with a simple comparison. For sample 1, I'm going to test the dye using the new primer, plus no aa-dUTP. I'm also going to do a positive control using a standard amplification using the old primers and aa-dUTP. Finally, I'll run a negative control using old primer and no aa-dUTP. I'll do this for both V4S3 and V6S3, since I should test both primer sets. | So the new primers are in. We ordered them a few weeks ago. All the primers have a 5' amino group, which should be compatible with the Alexa-dye coupling system. Thanks to [[Matt:LabNotes/2013-9-25|Matt's results]] yesterday, I know that when the dye coupling does not work, the Centri-Sep columns yield very little dye. So, before going nuts on making new probes, I'm going to test the water with a simple comparison. For sample 1, I'm going to test the dye using the new primer, plus no aa-dUTP. I'm also going to do a positive control using a standard amplification using the old primers and aa-dUTP. Finally, I'll run a negative control using old primer and no aa-dUTP. I'll do this for both V4S3 and V6S3, since I should test both primer sets. | ||
==Amino Primers Test== | |||
===qPCR=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#FFFF00" align="center" width="81" height="45" | | |||
| width="65" | 20 nM OligoPool | |||
| width="65" | 2X Kapa SYBR qPCR MM | |||
| width="65" | 100 uM AP1V41U | |||
| width="65" | 100 uM AP2V4 | |||
| width="65" | 100 uM AP1V61U | |||
| width="65" | 100 uM AP2V6 | |||
| width="65" | 50 mM aa-dUTP | |||
| width="65" | H20 | |||
| width="65" | Total Volume | |||
|- style="background-color:#8DB4E2;font-size:12pt" align="center" | |||
| height="15" | std 100 uL rxn | |||
| align="center" | 0.2 | |||
| align="center" | 50 | |||
| align="center" | 0.4 | |||
| align="center" | 0.4 | |||
| align="center" | 0.4 | |||
| align="center" | 0.4 | |||
| align="center" | 0.1 | |||
| align="center" | 46 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V4S3* (5x) | |||
| align="center" | 1 | |||
| align="center" | 250 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
| align="center" | 245 | |||
| align="center" | 500 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V6S3* (5x) | |||
| align="center" | 1 | |||
| align="center" | 250 | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 0 | |||
| align="center" | 245 | |||
| align="center" | 500 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S3 (5x) | |||
| align="center" | 1 | |||
| align="center" | 250 | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 0.5 | |||
| align="center" | 244.5 | |||
| align="center" | 500 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S3- (5x) | |||
| align="center" | 1 | |||
| align="center" | 250 | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 0 | |||
| align="center" | 245 | |||
| align="center" | 500 | |||
|} |
Revision as of 19:00, 26 September 2013
Amino Group Primers
So the new primers are in. We ordered them a few weeks ago. All the primers have a 5' amino group, which should be compatible with the Alexa-dye coupling system. Thanks to Matt's results yesterday, I know that when the dye coupling does not work, the Centri-Sep columns yield very little dye. So, before going nuts on making new probes, I'm going to test the water with a simple comparison. For sample 1, I'm going to test the dye using the new primer, plus no aa-dUTP. I'm also going to do a positive control using a standard amplification using the old primers and aa-dUTP. Finally, I'll run a negative control using old primer and no aa-dUTP. I'll do this for both V4S3 and V6S3, since I should test both primer sets.
Amino Primers Test
qPCR
20 nM OligoPool | 2X Kapa SYBR qPCR MM | 100 uM AP1V41U | 100 uM AP2V4 | 100 uM AP1V61U | 100 uM AP2V6 | 50 mM aa-dUTP | H20 | Total Volume | |
std 100 uL rxn | 0.2 | 50 | 0.4 | 0.4 | 0.4 | 0.4 | 0.1 | 46 | 100 |
V4S3* (5x) | 1 | 250 | 2 | 2 | 0 | 0 | 0 | 245 | 500 |
V6S3* (5x) | 1 | 250 | 0 | 0 | 2 | 2 | 0 | 245 | 500 |
V6S3 (5x) | 1 | 250 | 0 | 0 | 2 | 2 | 0.5 | 244.5 | 500 |
V6S3- (5x) | 1 | 250 | 0 | 0 | 2 | 2 | 0 | 245 | 500 |